PROJECT SUMMARY Two live, oral vaccines recommended by the World Health Organization (WHO) for childhood immunizations are oral rotavirus vaccines (ORV) and oral poliovirus vaccines (OPV). Both vaccines are implemented in the immunization programs of many middle and low income countries and are co-administered for programmatic reasons. Several studies have documented a significantly diminished immune response and vaccine take to ORV when OPV is co-administered, although there is limited interference of ORV on OPV vaccine response. There is limited data on interactions between the 2 vaccines in vitro and the mechanisms by which OPV interferes with ORV remain unknown. In this new grant application, we aim to identify: (i) how ORV and OPV co-infect and co-replicate in pediatric gastrointestinal epithelium, (ii) what innate immune effectors are produced in response to infections with both vaccines individually and together, and (iii) how these innate responses affect the replication of ORV and OPV. We hypothesize that innate intestinal epithelia cell responses to OPV and co-infection of two enteric vaccines in the pediatric gastrointestinal epithelium will reduce the replication of ORV and this factor contributes to the diminished immune response to ORV. We will use a biologically and physiologically relevant model of the human gastrointestinal epithelium called human intestinal enteroids (HIEs) for assessment of interactions and innate immune responses to OPV and ORV. HIE cultures developed from pediatric small intestinal biopsies will be used for infection with ORV and OPV strains that are currently being administered to children worldwide. Data generated from these studies will therefore be of direct relevance to the vaccines being studied and in age-groups where they are utilized. As investigators with expertise in epidemiology and field studies of enteric infections and vaccines, molecular virology and viral pathogenesis of poliovirus and rotavirus and in HIE culture, we will utilize the novel HIE technology to address a question of fundamental public health importance. The proposed study is designed to address both a co- infection effect that occurs when the vaccines are co-administered, and a prior-dose effect of OPV. The latter mimics the administration of a birth dose of OPV in many at-risk countries resulting in extended OPV shedding, possibly up to when a dose of ORV and OPV are co-administered. The establishment of a co-infection model for OPV and ORV interaction in pediatric human HIEs is critical to (i) gain mechanistic insights into vaccine virus interactions and innate immunity to live, attenuated vaccines in the gastrointestinal epithelium, (ii) develop physiologically relevant pre-clinical systems for evaluation of interventions to improve vaccine response before large, expensive clinical studies are carried out, and (iii) establish a platform that can be applied for assessment of interactions between vaccines and/or other enteric agents of clinical relevance.